Identification of Cancer Patients with Lynch Syndrome: Clinically Significant Discordances and Problems in Tissue-Based Mismatch Repair Testing

Home , Cancer syndrome, MLH1, MSH6, PMS2

Published OnlineFirst November 15, 2011; DOI: 10.1158/1940-6207.CAPR-11-0288

Cancer Prevention Research Article Research

Identiﬁcation of Cancer Patients with Lynch Syndrome: Clinically Signiﬁcant Discordances and Problems in Tissue-Based Mismatch Repair Testing

Angela N. Bartley1, Rajyalakshmi Luthra1, Devki S. Saraiya3, Diana L. Urbauer2, and Russell R. Broaddus1

Abstract Tissue-based microsatellite instability (MSI) analysis and immunohistochemistry for DNA mismatch repair proteins are accepted screening tools to evaluate patients with cancer for Lynch syndrome. These laboratory analyses are thus important tools in cancer prevention. Quality assurance review was conducted to identify test discordances and problems. These results were then analyzed in conjunction with genetic testing outcomes. Six hundred and forty-six consecutive tumors from 2002 to 2010 were examined. MSI-low tumors were excluded so that 591 tumors comprised the final analyses. Discordance was defined as a discrepancy between immunohistochemical and MSI analysis. Problem was defined as indeterminate or questionable immunohistochemical or MSI results. All results and clinical and family histories were centrally reviewed by two pathologists and one genetics counselor. Discordances and problems were identified in 23 of 591 (3.9%) of the tumors. Twelve of 102 MSI-high carcinomas (11.8%) and one of 489 microsatellite stable tumors had discordant immunohistochemistry. Of these 13 tumors, 11 were from patients who had personal and/or family cancer histories concerning for a germline mismatch repair gene mutation. In addition to discordances, 10 tumors with problematic immunohistochemical profiles were identified. Accurate evaluation of MSI was possible in all tumors. In summary, concordance between immunohistochemistry and MSI was high, particularly for tumors that are microsatellite stable. Greater frequency of test discordance was identified in the tumors that were MSI-high. Thus, a major consequence of the use of immunohistochemistry by itself as a screen is the failure to identify colorectal and endometrial cancer patients who likely have Lynch syndrome. Cancer Prev Res; 5(2); 320–7. 2011 AACR.

Introduction become important for the identification of patients with Lynch syndrome and has treatment and prognostic signifi- Defects in the DNA mismatch repair (MMR) system cause cance for patients with sporadic colorectal cancer. Screening replication errors or instability in DNA microsatellites. for Lynch syndrome can not only effectively prevent sub- Germline mutations in MMR genes MLH1, MSH2, MSH6, sequent cancers in the screened individual, but it can also and PMS2 cause the autosomal dominant hereditary disease prevent cancer development in relatives who also harbor a Lynch syndrome. More than 90% of tumors from patients germline mutation of an MMR gene. with this syndrome show high levels of microsatellite insta- Traditionally, clinical screening methods to help identify bility (MSI-high; ref. 1). MSI also occurs in 10% to 15% of families at risk for Lynch syndrome included the use of the sporadic tumors, especially colorectal and endometrial Amsterdam and Amsterdam II criteria. Because these criteria carcinomas, due to hypermethylation of the MLH1 pro- have been thought to lack sensitivity, particularly when an moter (2). Screening for defects in the MMR genes has extensive family history is unavailable, the revised Bethesda criteria were developed. The Bethesda criteria are more sensitive, focus on the patient medical history, and intro- Authors' Affiliations: Divisions of 1Pathology and Laboratory Medicine duced the category of MSI and tumor morphology into and 2Quantitative Sciences and 3Department of Surgical Oncology, Uni- clinical guidelines. However, these criteria do not address versity of Texas MD Anderson Cancer Center, Houston Texas patients with endometrial cancer and may fail to identify a Note: This original research was presented in part at the 100th United States large number of mutation carriers that do not meet the and Canadian Academy of Pathology Annual Meeting, February 27 to March 4, 2011, San Antonio, TX. clinical criteria (3). To further help identify individuals at risk for Lynch syndrome, several clinical prediction models Corresponding Author: Russell R. Broaddus, Department of Pathology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe have been developed to quantify the risk for germline MMR Blvd. Houston, TX 77030. Phone: 713-795-2794; Fax: 713-792-5532; mutations (4–6). These have comparable sensitivities to the E-mail: [email protected] Bethesda criteria and can provide quantitative risk assess- doi: 10.1158/1940-6207.CAPR-11-0288 ment (7). However, such prediction models require subse- 2011 American Association for Cancer Research. quent genetic testing and are not helpful in identifying

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Molecular Analyses in Mismatch Repair Testing

patients with sporadic MSI-high colorectal cancers requir- Molecular analyses ing alternative treatment regimens. All immunohistochemical and PCR-based molecular Two tissue-based screening methods used to assess analyses were conducted in Clinical Laboratory Improve- tumors for MMR defects include PCR-based MSI testing ment Amendments (CLIA)- and College of American and immunohistochemistry. In general, a number of large Pathology–approved laboratories. Immunohistochemistry studies have concluded that MSI and immunohistochem- was carried out for DNA MMR gene products MLH1 (G168- istry have comparable sensitivity and specificity in detecting 15, 1:25; BD Biosciences Pharmingen), MSH2 (FE11, 1:100; MMR defects (8–12). Because it is generally associated with Calbiochem), MSH6 (44, 1:300; BD Biosciences Pharmin- lower cost, faster turnaround time, and wider availability in gen), and PMS2 (Alb-4, 1:125; BD Biosciences Pharmingen) smaller pathology laboratories, some advocate that immu- as previously described (18). Tumors showing loss of nohistochemistry could be used as the sole method of nuclear MLH1, MSH2, PMS2, or MSH6 were classified as screening in certain cancers (9, 13–16). negative for the protein expression of each respective mark- At our institution, we conduct both PCR-based MSI and er. Stromal and normal tissues within the tumor served as immunohistochemistry concurrently. Immunohistochem- internal positive controls. ical–MSI analysis discordances have been acknowledged in MSI analysis was conducted using 6 National Cancer the research setting, but the frequency and nature of these Institute (Bethesda, MD) recommended microsatellites problems have not been formally examined in a quality (19) with the addition of analysis for the mutational inac- assurance fashion in the clinical laboratory. In surgical tivation of TGF-b receptor type II (TGFBR2). Therefore, 7 pathology practice, correlation reviews (cytology/histology microsatellites comprise the MSI panel. A tumor was des- and frozen section final pathology correlations) are com- ignated as MSI-high if it showed allelic shift at 3 or more monly used tools for quality assurance (17). This type of markers and as MSI-low with allelic shift in one or 2 quality assurance review has not been previously applied to markers. Those without allelic shifts were designated micro- the tissue-based approach to MMR testing. Therefore, we satellite (MS)-stable. A methylation-specific PCR MLH1 examined sources of discordances and problems in tissue- promoter methylation assay and BRAF V600E mutational based testing to help optimize this approach in our patient testing by pyrosequencing were conducted on a subset of population. cases (20, 21).

Materials and Methods Statistical analysis It is controversial whether MSI analysis or immuno- Patient population and study design histochemistry represents the "gold standard" when con- After obtaining Institutional Board approval, we sidering the tissue testing approach. Therefore, tradition- obtained the results and clinical records for all cancer al measures of testing efficacy, such as sensitivity and patients who had MSI and/or immunohistochemical test- specificity, are likely not appropriate here. Instead, the ing between August 2002, and August 2010, at the Uni- measures calculated were concordance (percentage of versity of Texas MD Anderson Cancer Center (Houston, pairs in agreement) and discordance (percentage of pairs TX; n ¼ 736 patients). For 107 of these patients, only in disagreement). Specifically, we calculated concordance immunohistochemical or MSI analysis had been previ- and discordance and their exact 95% confidence intervals ously conducted, so these patients are excluded from (CI) for MSI-high tumors, MS-stable tumors, tumors further consideration. Only those patients with both MSI with loss of an MMR protein by immunohistochemistry, testing and immunohistochemical results for the same and tumors with retained expression of all 4 MMR tumor were included (n ¼ 629). Demographic, clinical, proteins. and pathologic data were collected. Initial immunohistochemical slides were examined by a group of 14 dif- Results ferent gastrointestinal and gynecologic pathologists, whereas initial MSI analyses were evaluated by 7 gastro- Patient tumor population intestinal pathologists and 5 molecular pathologists. Dis- From August 2002 to August 2010, 629 patients had cordancewasdefinedasadiscrepancybetweentheresult tumors analyzed by both immunohistochemical and MSI of MSI and immunohistochemistry. Problem was defined analysis. Twenty-six of these patients had more than one as a case with indeterminate or questionable immuno- tumor analyzed. Therefore, there are a total of 646 tumor histochemical or MSI results. The immunohistochemical analyses included in this study. The majority of tumors were slides and the MSI chromatograms for all discordant/ colorectal adenocarcinomas (88%), followed by endome- problematic cases were rereviewed by 2 pathologists trial carcinomas (7%; Table 1). Table 2 summarizes the (R.R. Broaddus and A.N. Bartley) and results and patient distribution of tumors by MSI status. There were 102 histories discussed with a genetics counselor (D.S. Sar- (15.8% of the total) MSI-high carcinomas [78/102 colorec- aiya). Any additional testing conducted, including MLH1 tal (76%), 17/102 endometrial (17%), and 7/102 other methylation, BRAF mutational analysis, and subsequent (7%)] and 55 patients had MSI-low carcinomas [49/55 genetic sequencing and large rearrangement testing for colorectal (89%), 4/55 endometrial (7%), and 2/55 other the 4 most common MMR genes, was also documented. (4%)]. The remaining 489 tumors were MS-stable.

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Table 1. Tumor analyses (total number of carcinoma with immunohistochemical loss of MLH1 and PMS2) underwent germline testing; 4 of 9 (44.4%) were tumors analyzed ¼ 646) identified to have MMR gene alterations. One of these alteration positive patients (patient 3) has an MLH1 Tumor type % total Median mutation that is known to be pathogenic. The remaining (number of patient age, identified gene alterations are variants of undetermined tumors) y (range) significance. Patient 7 had an in-frame duplication of Colorectal adenocarcinoma 88 (568) 59 (18–88) exons 3 to 5 of MLH1. This duplication is not predicted Endometrial carcinoma 7 (45) 55 (19–85) to result in protein truncation, but it is possible that this Small bowel adenocarcinoma 2 (13) 44 (29–59) would affect protein function. Of the 489 MS-stable Colorectal adenoma 1 (7) 45 (42–56) tumors, only one had loss of an MMR protein by immu- Ovarian carcinoma 0.5 (3) 57 (45–72) nohistochemistry. This was an endometrial carcinoma Pancreatic adenocarcinoma 0.5 (3) 64 (61–69) with loss of MLH1 and PMS2 by immunohistochemistry Other 1 (7) 40 (38–55) andnoevidenceofMLH1 methylation. No mutations of MLH1 were detected on subsequent sequencing or large rearrangement analysis. The patient declined genetic test- PMS2 Immunohistochemistry–MSI agreement ing for and is now deceased. Table 3 summarizes the concordances and discordances Fifty-three of 55 MSI-low tumors had positive immuno- between immunohistochemical and MSI analysis for the histochemical expression of MLH1, MSH2, MSH6, and MS-stable and MSI-high tumors (n ¼ 591). MSI-low tumors PMS2. None of these patients had further genetic testing. (n ¼ 55) were specifically excluded from these analyses. The One 53-year-old patient with an MSI-low rectal adenocar- nature of MSI-low tumors is controversial and not well cinoma had loss of MSH6 by immunohistochemistry; sub- MSH6 understood, so it is not possible to determine whether sequently, a deleterious mutation was discovered. A immunohistochemical and MSI analyses for individual second 68-year-old patient with an MSI-low rectal adeno- patients are concordant. Overall, for the MS-stable and carcinoma had immunohistochemical loss of MSH2 and MSH2 MSI-high tumors, the agreement between immunohisto- MSH6, suggestive of mutation. This patient declined chemical and MSI analysis was quite good (97.80% con- genetic testing to confirm this, however. cordance, exact 95% CI, 96.27–98.82). The highest level of agreement occurred in MS-stable tumors with intact expres- Problems—immunohistochemistry sion of all 4 MMR proteins. The lowest level of concordance Problems were identified in 10 patients. These problems occurred in MSI-high tumors with loss of at least one MMR do not overlap with the discordances presented in Table 4. n ¼ protein by immunohistochemistry. The problems were grouped as selection error ( 2), Discordances were identified in 13 of 591 (2.2%) pathologist error in immunohistochemical interpretation n ¼ tumors with both MSI and immunohistochemical testing. ( 6), or unusual pattern of immunohistochemical n ¼ A detailed summary of these discordances is presented expression ( 2). Selection error involved 2 different in Table 4. Twelve of 102 (11.8%) MSI-high tumors had patients, each with 2 separate primary colorectal adenocar- discordant immunohistochemical tumor profiles, show- cinomas. The proximal tumor in both was MS-stable with ing intact immunohistochemical expression of MLH1, intact nuclear expression for all 4 MMR proteins by immu- MSH2, MSH6, and PMS2 proteins. Seven of 12 tumors nohistochemistry. The distal tumor in both patients was were from patients who were younger than 50 years at age subsequently tested and found to be MSI-high with immu- of diagnosis, and 8 of 12 tumors were from patients who nohistochemical loss of MSH2 and MSH6. One of these had a first-degree relative with a Lynch syndrome–asso- patients agreed to further genetic testing and was found to MSH2 ciated cancer. Following genetic counseling, 9 patients (8 have an mutation. patients with MSI-high tumors but retained immunohis- Six colorectal adenocarcinomas had pathologist difficul- tochemical expression of MLH1, MSH2, MSH6, and ty/error in immunohistochemical interpretation. For 2 of PMS2 and the one patient with MS-stable endometrial these, our central review changed the immunohistochemical result. The first tumor with pathologist error was from a 32-year-old with an MSI-high rectal adenocarcinoma that was initially interpreted as having positive expression for all Table 2. Distribution of tumor analyses by MSI ¼ 4 MMR proteins. Rereview showed that the tumor had status (total number of tumors analyzed 646) diffuse, strong expression for MLH1, MSH2, and PMS2. Most of the tumor cells, however, were entirely negative for % total MSH6. A few tumor cells had faint nuclear expression for MSI status (number of tumors) MSH6 that was much weaker than that of adjacent stromal MSI-high 15.8 (102) cells and normal mucosa (Fig. 1). This patient was subse- MS-stable 75.7 (489) quently identified to have an MSH6 mutation. The second MSI-low 8.5 (55) tumor with pathologist error was a rectal adenocarcinoma that was MS-stable and initially interpreted as negative for

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Table 3. Summary of concordances and discordances between immunohistochemical and MSI analysisa

N (%) 95% CI

MSI-high (N ¼ 102) IHC shows loss of expression of at least one MMR protein 90 (88.24) 80.35–93.77 IHC shows intact expression of all 4 proteins 12 (11.76) 6.23–19.65 MS-stable (N ¼ 489) IHC shows loss of expression of at least one MMR protein 1 (0.20) 0.01–1.13 IHC shows intact expression of all 4 proteins 488 (99.80) 98.87–99.99 IHC—loss of at least one MMR protein (N ¼ 91) MSI-high 90 (98.90) 94.03–99.97 MS-stable 1 (1.10) 0.03–5.97 IHC—intact MMR protein expression (N ¼ 500) MSI-high 12 (2.40) 1.25–4.15 MS-stable 488 (97.60) 95.85–98.75 Overall agreement IHC shows loss of expression of at least one MMR protein 578 (97.80) 96.27–98.82 and MSI-high or IHC shows intact expression of all 4 proteins and MSI-stable IHC shows loss of expression of at least one MMR protein and 13 (2.20) 1.18–3.73 MSI-stable or IHC shows intact expression of all 4 proteins and MSI-high

Abbreviation: IHC, immunohistochemistry. aThis table summarizes the results for n ¼ 591 tumor analyses, including MSI-high tumors (n ¼ 102) and MS-stable tumors (n ¼ 489). Tumors that were MSI-low were speciﬁcally excluded from this analysis, because of the uncertainty of what represents immunohistochemical/MSI concordance/discordance in this group.

MLH1 by immunohistochemistry. Re-review revealed that PMS2. Methylation of MLH1 was detected in this tumor, so the background stromal cells were also negative for MLH1 this was interpreted as a sporadic endometrial carcinoma. and that the immunostaining had, therefore, not worked The second unusual pattern was an MSI-high colorectal properly. Immunohistochemistry was repeated using a dif- adenocarcinoma with strong expression of MLH1 and ferent tumor block and showed convincing MLH1 protein MSH2 and complete loss of MSH6 and PMS2 (Fig. 3). These expression in the tumor (Fig. 2). The remaining 4 tumor tests were subsequently repeated twice with rereview of each analyses in this category involved difficulties with MSH6 set with the same result. This patient did not assent to immunohistochemistry. Two of these were MSI-high carci- genetic testing. nomas (one colorectal, one endometrial) in which the tumors were positive for MLH1, MSH2, and PMS2, but the Problems—MSI MSH6 immunohistochemistry did not work (internal con- Given the intense scrutiny of the immunohistochemical trol benign tissue also did not stain). Both of these were tests summarized earlier, we next reviewed the results of all from patients who had a family history of a Lynch syn- 646 tumor analyses for problems in DNA amplification in drome–associated cancer but neither fulfilled Amsterdam the MSI analysis to provide better balance to the quality criteria. Neither of these patients consented to further assurance review. Overall, only 0.6% of the microsatellites genetic testing. The last 2 tumors in this category were amplified suboptimally. No tumor had more than one MS-stable colorectal adenocarcinomas which had strong, failed microsatellite amplification. Thus, accurate determi- diffuse nuclear expression for MLH1, MSH2, and PMS2, but nation of MSI status was possible for all 646 tumor analyses. the MSH6 expression in the tumor was initially interpreted Only 5 microsatellites, or 1% of the total tested, for the as only focal or weak. However, upon our rereview, the tumors in the MSI-high category failed to amplify. This MSH6 immunohistochemistry was interpreted as positive included one each in 5 different tumors. This did not pose a (retained expression), so these patients did not undergo diagnostic problem as the remaining 6 microsatellites did genetic testing. amplify. For the 55 MSI-low tumors, only 6 of 385 micro- Two tumors with unusual immunohistochemical stain- satellites (2%) tested failed to amplify (one each in 6 ing patterns were identified. One unusual staining pattern different tumors). Even if this failed microsatellite was in an MS-stable endometrial adenocarcinoma showed actually unstable, this would not be sufficient to redesignate strong nuclear staining for MSH2 and MSH6, but the tumor the tumor as MSI-high, as instability in at least 3 of 7 had definite positive foci and definite negative foci for both microsatellites is required for a tumor to be MSI-high. For MLH1 and PMS2. In the negative foci, internal positive the 489 MS-stable tumors, only 13 of 3,423 microsatellites control stromal cells retained expression of both MLH1 and (0.4%) failed to amplify, one each from 13 different tumors.

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Table 4. Summary of tumors with discordant MSI and immunohistochemical results

Immunohistochemistry

S.No MLH1 MSH2 MSH6 PMS2 MSI MLH1 Germlinea BRAF Tumor Age AC/FH status methylation alteration mutation location

1 þþþþMSI-H Neg MSH2 Neg Rectum 48 Neg/no 2 þþþþMSI-H NP NP NP Colon 47 Neg/no 3 þþþþMSI-H Neg MLH1 NP Colon 62 Pos/yes 4 þþþþMSI-H NP MLH1, PMS2 NP Colon 57 Neg/no 5 þþþþMSI-H Present NP NP Small bowel 59 Neg/no 6 þþþþMSI-H NP Neg NP Colon 47 Neg/yes 7 þþþþMSI-H Neg MLH1 Neg Colon 29 Pos/yes 8 þþþþMSI-H NP Neg NP Colon 43 Neg/yes 9 þþþþMSI-H Neg Neg Neg Uterus 51 Neg/yes 10 þþþþMSI-H Neg Neg Neg Colon 63 Neg/yes 11 þþþþMSI-H Neg NP Neg Uterus 32 Pos/yes 12 þþþþMSI-H Neg NP Neg Colon 47 Neg/yes 13 þþMSS Neg Neg Neg Uterus 85 Neg/yes

Abbreviations: MSI-H, MSI-high; MSS, microsatellite stable; NP, not performed; Neg, negative; Pos, positive; AC, Amsterdam II criteria; and FH, family history of a Lynch syndrome–associated cancer. aThe MLH1 mutation [618del (1852del3)] in patient 3 is known to be pathogenic. This is an in-frame deletion of 3 nucleotides that results in the omission of lysine from amino acid position 618 but does not lead to premature protein termination. The germline alterations in patients 1, 4, and 7 are currently classified as variants of undetermined significance. For patient 1, there is a point mutation (T677R) that substitutes arginine for threonine at 677. For patient 4, 3 different mutations were identified, one in PMS2 [PMS2 c.1437C>G (p.His479Gln)] and 2 in MLH1 [MLH1 c.290A>G (p.Tyr97Cys) and MLH1 c.299G>A (R100Q, Arg100Gln)]; none of these are currently predicted to result in protein truncation. Patient 7 had an in-frame duplication of exons 3 to 5 of MLH1. This is not predicted to cause protein truncation, but it possible that the duplication would affect protein function. Patients 6 and 10 had negative MLH1, MSH2, and MSH6 germline testing. Patient 8 had negative MLH1, MSH2, MSH6, and PMS2 germline testing. Patient 9 had negative MSH6 germline testing. Patient 13 had negative and MLH1 and MSH2 germline testing and declined further testing.

Discussion 11.8% of the MSI-high carcinomas had intact immunohistochemical expression of MLH1, MSH2, MSH6, and PMS2. Because immunohistochemistry is less expensive, more A previous population-based examination of 500 unselect- widely available, relatively simple to carry out, and thought ed colon cancer patients showed a comparable percentage to be comparable with MSI analysis, a number of studies (9.7%) of MSI-high cancers with intact immunohistochem- have advocated the use of immunohistochemistry alone in ical expression of MMR proteins (23). Importantly, our the initial evaluation for MMR defects (14–16, 22). Indeed, study suggests that a signiﬁcant number of possible patients we conﬁrm that, in general, concordance between immu- with Lynch syndrome would not be detected if only immunohistochemical and MSI analysis is quite high. However, nohistochemistry was used as a screening technique. When

A B C

Figure 1. Immunohistochemistry in an MSI-high colorectal adenocarcinoma initially interpreted as weakly positive for MSH6 by immunohistochemistry. A, the tumor and adjacent normal crypts have strong MLH1 nuclear expression (100). B, MSH6 is negative in the tumor cells but strongly positive in adjacent benign crypts and stromal cells (100). C, higher magniﬁcation of MSH6 immunohistochemistry with slight nuclear blush of staining in neoplastic cells. This staining is far lighter than that of stromal internal controls or adjacent nonneoplastic crypts (200).

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Figure 2. Immunohistochemistry for an MS-stable colorectal A B adenocarcinoma initially interpreted as negative for MLH1 (A and B, 200). A, original MLH1 slide shows that both neoplastic glands and adjacent stromal cells are negative for MLH1. This is a suboptimal immunohistochemistry test that cannot be adequately interpreted. B, repeat MLH1 immunohistochemistry using a different parafﬁn block of the same tumor shows strong MLH1expression in both the tumor and adjacent stromal cells.

family history and the molecular diagnostics tests are due to young age of cancer onset or family history of a Lynch considered together, only one (#5) of the 12 MSI-high syndrome–related cancer. Reﬂex testing of all patients with carcinomas with intact MMR immunohistochemistry sum- colorectal cancer did not begin at our institution until marized in Table 4 is clinically thought to be sporadic. This September, 2009. Previously, Thibodeau and colleagues percentage of likely Lynch syndrome cases is relatively high, showed that, in a large series of approximately 1,400 especially compared with that of other population-based patients with colorectal cancer, there are more immuno- screening studies (23). This higher percentage may be due to histochemical–MSI discrepancies when the patient popu- the fact that the vast majority of the patients in our study lation screened had moderate to high risk for Lynch were initially clinically suspected as having Lynch syndrome syndrome (24). Interestingly, in our current series, we found

A B

Figure 3. Unusual immunohistochemical staining pattern in an MSI-high colorectal adenocarcinoma positive for MLH1 (A) and MSH2 (B) and negative for PMS2 (C) and MSH6 (D). For PMS2 and MSH6, note the presence of C D nuclear staining in the adjacent benign stromal cells, indicating that the immunohistochemical tests are working appropriately. A–D, 200.

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that 7 of 13 of our immunohistochemical–MSI discor- of microsatellite DNA to amplify for adequate testing, were dances were prior to September 2009, and 6 of 13 were also identified in our study. Of the 646 tumors reviewed, no after this date. So, approximately half of the discordances tumor had more than one microsatellite fail PCR amplifi- occurred after commencement of universal screening for cation. Given that a panel of 7 microsatellites is used, rather colorectal cancer. than just one or 2, the failure of one microsatellite to amplify In the present study, 12 patients with cancer had MSI- still allows for distinguishing MSI-high from MS-stable. high tumors but intact MMR protein expression by immu- Although our quality assurance review identified more nohistochemistry. Of these 12, 8 agreed to genetic testing. shortcomings in immunohistochemistry-based testing, it Genetic testing in these patients is problematic, as the should be emphasized that immunohistochemistry plays immunohistochemical results cannot help to direct the an important role in the evaluation of patients with MSI- testing. Seven of these 8 were suspicious for Lynch syn- high carcinomas and can help direct future germline testing. drome based on lack of tumor MLH1 methylation, lack of The identification of immunohistochemical loss of MSH2, BRAF mutation, young age of cancer onset, and/or presence MSH6, or PMS2 in an MSI-high tumor is virtually diagnostic of Amsterdam criteria/family history of a Lynch syndrome for Lynch syndrome, even when germline testing fails to cancer. Missense mutations could potentially lead to full- reveal a mutation. These patients and their relatives should length, but nonfunctional, MMR proteins, which would still be offered cancer prevention services at an earlier age. help to explain the immunohistochemical–MSI discor- Considering the number of possible patients with Lynch dances. However, only 1 of these 8 (12.5%) patients had syndrome who would be missed by screening by immuno- a pathologic MMR mutation detected (Table 4, #3, MLH1 histochemistry alone, we cannot recommend immunohis- mutation). The remaining 7 patients either had variants of tochemistry as the sole tissue-based screening technique. undetermined significance or no germline variation Rather, for population-based screening of patients with detected. Tumor 7 was from a patient with an MLH1 in- colorectal cancer, it might be preferred to use MSI analysis frame duplication of exons 3 to 5; although this duplication as an initial screen and then carry out immunohistochem- is not thought to cause protein truncation, it could possibly istry in MSI-high cases, as recommended by others (12, 25– affect protein function. In our previous studies examining 27). Alternatively, MSI and immunohistochemistry can be patients with endometrial cancer younger than 50 years of initiated concurrently in patients with moderate or high risk age (20), 11 patients had MSI-high endometrial cancers of Lynch syndrome (24). Of course, it is imperative that with loss of an MMR protein detected by immunohis- patients have access to genetic counseling and subsequent tochemistry. From these 11 patients, we detected 9 patho- germline mutation analysis if these tissue-based tests are logic mutations (81.8%). Three more patients with endo- informative. metrial cancer had MSI-high tumors but intact MMR pro- The tissue-based testing of patients with endometrial tein expression by immunohistochemistry; for these 3 cancer is more problematic. Endometrial cancers and colon patients, no mutations were identified by genetic testing. cancers from the same patient can exhibit different levels of Collectively, these results suggest that when immunohis- MSI, with the endometrial cancers often being MSI-low or tochemistry detects loss of expression of an MMR protein, MS-stable (28). MSH6 mutation carriers are especially there is a higher likelihood of ultimately detecting a path- prone to developing endometrial cancer, and these tumors ologic MMR gene mutation. Conversely, for tumors that are are not typically MSI-high (29, 30). Experience with MSI MSI-high but have retained positive expression of MMR analysis is even more limited with other cancer types asso- proteins by immunohistochemistry, there could be a lower ciated with Lynch syndrome, such as cancers of the ureter, chance of ultimately discovering a pathologic Lynch syn- ovary, and small intestine. Thus, concurrent MSI analysis drome mutation. The reasons for this at this time are not and immunohistochemistry should be conducted in clear. Given time and more experience, it is possible that patients with these extracolonic malignancies. some of these identified variants of undetermined signifi- fl cance become reclassified as pathologic mutations. Or, Disclosure of Potential Con icts of Interest mutations in less common genes regulating DNA MMR D.S. Saraiya is currently employed by Myriad Genetic Laboratories, Inc., as may be responsible for the high levels of MSI. It is also a Regional Medical Specialist (Certified Genetic Counselor). However, all possible that alternative, not yet identified, genetic mechan- content and writing for the manuscript were completed while she was an isms other than MMR are generating tumor MSI. employee of MD Anderson Cancer Center. No potential conflicts of interest were disclosed by the other authors. In addition to the discordances, our quality assurance review identified several different problems in the tissue- Grant Support based approach, particularly with immunohistochemistry. Several of these problems are readily correctable, particu- The study was supported by NIH 2P50 CA098258-06 SPORE in Uterine Cancer (R.R. Broaddus) and NIH CA016672 Cancer Center Support Grant. larly the recognition of lack of internal positive control The costs of publication of this article were defrayed in part by the staining and the testing of all tumors in patients with payment of page charges. This article must therefore be hereby marked synchronous primaries. Other problems, such as interpre- advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tation of weak or focal immunohistochemical staining (Fig. 1), may require considerable experience to accurately Received June 2, 2011; revised November 2, 2011; accepted November 7, interpret (25). Problems in the MSI analysis, chiefly failure 2011; published OnlineFirst November 15, 2011.

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Molecular Analyses in Mismatch Repair Testing

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www.aacrjournals.org Cancer Prev Res; 5(2) February 2012 327

Identification of Cancer Patients with Lynch Syndrome: Clinically Significant Discordances and Problems in Tissue-Based Mismatch Repair Testing

Angela N. Bartley, Rajyalakshmi Luthra, Devki S. Saraiya, et al.

Cancer Prev Res 2012;5:320-327. Published OnlineFirst November 15, 2011.

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